1. Field of the Invention
The present invention relates to a method for separating nucleated fetal red blood cells and other nucleated fetal cells from maternal blood using a triple gradient gel and antibody panning.
2. Discussion of the Background
The examination of fetal cells for early detection of fetal diseases and genetic abnormalities is undertaken in approximately one out of every thirty pregnant women. The main indication is maternal age (over 35 years). The tests may involve DNA gene typing or, more commonly, the use of live fetal cells for chromosomal karyotyping.
Fetal cells are usually obtained by amniocentesis, the removal of amniotic fluid from the amniotic cavity within the amniotic sac or placenta. The procedure presents a risk of harm to the fetus, particularly after the first trimester of pregnancy. The risk to the fetus together with the high cost of the procedure have prevented the establishment of examination of fetal cells for early detection of abnormalities as a routine procedure in pregnancy.
In the late 1970's and early 1980's, Herzenberg and his colleagues reported that fetal cells were present in maternal blood as early as 15 weeks gestation. Maternal and fetal cells were separated using fluorescence-activated cell sorting (FACS) by staining maternal blood for a distinguishing paternal HLA antigen. To date, the technique has not been successfully adapted for use as a clinical technique for either karyotyping or fetal DNA analysis.
Fetal cells present in maternal blood have been used to perform analysis of genes present in the fetus. In one technique, the maternal and fetal cells were not separated and the DNA from the cell mixture is amplified with Y chromosome-specific primers to determine whether the fetus is male. It has been suggested that DNA amplification techniques can also be performed to detect gene sequences associated with disease in this manner. Of course, the method cannot be used where the mother is a carrier for the trait.
To date, amniotic fluid has been the only source of antenatal cells to provide a sufficient number of live cells for karyotyping. Furthermore, DNA analysis methods have only been possible in relatively limited situations which depend on particular differences in maternal and fetal cells, e.g. presence of the Y chromosome in the fetus or presence of HLA-A2 antigen on fetal, but not maternal, cells.
Herzenberg and his colleagues have described methods for separating maternal and fetal cells in maternal blood using fluorescence-activated cell sorting (FACS). In Herzenberg et al, Proc. Natl. Acad. Sci. USA 76:1453-1455 (1979), cells in blood samples from 15-week pregnant HLA A2-negative women were stained for HLA A2 antigen. Stained cells were separated by FACS and collected to enrich the population of fetal cells. Although the technique was demonstrated to effectively identify male, HLA A2-positive cells in maternal blood, to date the technique has not been successfully adapted for general applicability.
In Iverson et al, Prenat. Diag. 1:61-73 (1981), peripheral blood lymphocytes (PBLs) from either 15 week or 21 to 25 week pregnant women were examined. If the woman was HLA A2-negative, her cells were stained with anti-HLA A2 reagents, sorted by FACS onto microscope slides (for fetuses who were HLA A2-positive), stained with quinacrine and examined microscopically for Y chromatin-positive cells.
Bianchi et al, Cytometry 8:197-202 (1987) report a technique that allows direct hybridization to the DNA of cells which were flow sorted onto nitrocellulose filters which eliminates the need for a DNA isolation step. The method was performed on human cord blood. The technique is reportedly useful in situations where there is a limited amount of DNA available for analysis such as for fetal cells recovered from maternal blood.
U.S. Pat. No. 4,675,286 describes a method for obtaining fetal cells for diagnostic examination in which detached cells from the uterine cavity and outer surface of the amniotic sac are incubated with a separation antibody which binds preferentially to either fetal or maternal cells. The antibody can be bound to an insoluble support or conjugated with a fluorescent label and removed with a cell sorter to effect separation.
Albright et al, Cytometry 7:536-543 (1986) describe the use of centrifugal separation of cells in sputum specimens as an alternative to flow cytometry.
Society for Clinical Cytology 1988 Abstracts p.6 describe in situ hybridization for detection of structural and numerical abnormalities using nonradioactive probes for detection of aneuploidy and translocations (Pinkel et al, No. 13). Commercial probes are available for chromosomes 13, 18 and 21.
Density gels and centrifugation are routinely used to separate certain populations or subpopulations of blood cells from adult blood. A variety of these methods including use of a one step density gradient are summarized in U.S. Pat. No. 4,255,256, for example. In these methods, the cell mixture is placed on the surface of a gel in a tube, and the components are settled into layers by gravity or centrifugation. Single density gradient gels generally yield two zones, one at the surface of the gel and the other at the bottom. Continuous density gradient gels yield zones of cells throughout the gel separated by density. The patent also describes discontinuous density gradients with two or more separating solutions of different densities layered on top of one another. The densities are selected to provide a discontinuous gradient over a desired range. In the procedures, nucleated cells such as lymphocytes are usually collected as a mixture separated from mature red blood cells.
Each of the above-described references and the publications cited therein are hereby incorporated by reference into this application.